1. Field of the Invention
The present invention pertains to the preparation of humanized antibodies, which are capable of binding to free IgE, membrane-bound IgE on B lymphocytes, IgE bound by CD23, but not to IgE bound by high-affinity IgE.Fc receptors on mast cells. The invention also pertains to the therapeutic applications of such antibodies in treating IgE-mediated diseases, including allergic asthma, allergic rhinitis, atopic dermatitis, food allergy, chronic spontaneous (idiopathic) urticaria, chronic rhinosinusitis, systemic mastocytosis, cutaneous mastocytosis, allergic bronchopulmonary aspergillosis, recurrent idiopathic angioedema, and eosinophil-associated gastrointestinal disorder.
2. Description of Related Art
Allergy is a hypersensitive state induced by an exaggerated immune response to a foreign, harmless antigen. An immediate hypersensitivity response occurs through the interaction of immunoglobulin E (IgE) and the high-affinity IgE.Fc receptor (FcεRI) present on the surface of mast cells and basophils in the presence of allergen which is capable of reacting with the FcεRI-bound IgE, causing the release of performed and newly-synthesized mediators from these inflammatory cells. Most allergic diseases are IgE-mediated. It is well known that a number of non-allergic diseases, which do not involve an immune response to a foreign antigen, particularly inflammatory skin diseases, are also IgE-mediated.
There are two major receptor for IgE, FcεRI and the low-affinity IgE.Fc receptor FcεRII (also referred to as CD23). FcεRI is predominantly expressed on the surface of mast cells and basophils in humans, where it is a tetrameric complex consisting of one α-chain, one β-chain, and two disulfide-bound γ-chains. Activation of FcεRI on mast cells and basophils by allergens leads to degranulation, while activation of FcεRI on dendritic cells leads to IgE-mediated allergen presentation.
CD23 is a type II transmembrane glycoprotein of approximately 45 kDa molecular weight comprising a C-type lectin-like domain, followed by a stalk region, which bears several repeats that serve as a putative leucine zipper to form coiled-coil trimers, a single membrane-spanning region, and a short N-terminal cytoplasmic domain. Indeed, the affinity of monomeric CD23 to IgE (KD=10−6-10−7 M) is much lower than that of FcεRI (KD=about 10−10 M), but substantially increased in its trimeric form (KD=10−8-10−9 M). The FCER2 gene encoding CD23 polypeptide possesses 2 alternative transcription start sites driving synthesis of two mRNA variants which leads to produce two CD23 isoforms differing in the first seven (CD23a) and six (CD23b) amino acids of the N-terminal cytoplasmic tail. CD23a is exclusively and contiguously expressed by B cells, whereas CD23b is induced by IL-4 on the surface of monocyte/macrophage, B cells, T cells, eosinophils, dendritic cells, and epithelial cells. CD23 on B cells is thought to contribute to regulation of IgE production and antigen presentation, while CD23 on macrophages and dendritic cell is involved in phagocytosis/endocytosis, clearance of antigen-IgE complex and antigen presentation. Additional function of CD23 on epithelial cells includes the transportation of IgE. The antigen-IgE complex directly penetrate across the epithelium to the lumen space and vice versa. CD23 can also be released from cell surfaces as a range of free soluble CD23 (sCD23) proteins of 37, 33, 25, and 16 kDa. The predominant metalloprotease responsible for CD23 shedding in vivo is ADAM10 gene, which generates 37 kDa or 35 kDa sCD23 species in a trimeric form. A further naturally occurring sCD23 is derCD23 produced by action of the Der p1 protease found in the faeces of the house dust mite, dermatophagoides pteronyssinus. The cleavage of CD23 by Der p1 yields the 16 kDa monomeric derCD23. It is shown that trimeric sCD23 fragments were observed to be key molecules for potentiating spontaneous IgE synthesis, whereas the smaller monomeric sCD23 appears to down-regulate IL-4 stimulated IgE synthesis.
IgE exists in a secreted form and a membrane-bound form, which appears to be splicing variants. The constant regions of the ε chain of secreted form of IgE harbors CH1-CH2-CH3-CH4 domains, whereas the ε chain of membrane-bound form of IgE having a membrane-bound IgE (mIgE) is found in two isoforms as a result of alternative splicing in humans. The ε chain of both isoforms of human mIgE contains a migis-ε and a membrane-anchoring peptide. One isoform contains only the migis-ε peptide (15 amino acid long) between the CH4 domain and the membrane-anchoring peptide (referred to as “short form”), whereas the second isoform additionally contains an extra 52 amino acid long domain (referred to as CεmX domain) between the CH4 domain and the migis-ε peptide (referred to as “long form”).
Since IgE plays a central role in mediating most allergic diseases, several strategies have been proposed to control IgE levels in the body or to regulate IgE synthesis, such as anti-IgE, anti-IL-4/IL-13 and anti-CD23. Omalizumab (Xolair®) is a recombinant humanized monoclonal anti-IgE antibody that binds to free IgE circulating in the serum and membrane-bound IgE on B cells but not FcεRI-bound and CD23-bound IgE on cell surface. Omalizumab causes significant (up to 99%) reduction of serum free IgE in allergic patients, leading to inhibit IgE binding to FcεRI and subsequently a down-regulation of FcεRI on basophils and mast cells. Numerous clinical trials in many IgE-mediated diseases, such as allergic asthma, chronic urticaria, allergic rhinitis, atopic dermatitis, etc., have shown that Omalizumab is efficacious and safe in treating these diseases. Omalizumab has been approved for treating severe allergic asthma and chronic idiopathic urticaria in many countries. Moreover, lumiliximab, which is an anti-CD23 monoclonal antibody consisting of primate (cynomolgus macaque) variable regions and human constant regions, is found to bind to the C-type lectin domain preventing IgE from binding to CD23, thus leading to stabilization of surface CD23 and reduction of the proteolytic cleavage of CD23. Lumiliximab has been reported to inhibit the germline ε transcripts, decrease the IgE production of human peripheral blood mononuclear cell (PBMCs) in culture, and reduce blood IgE levels in allergic patients.
In a 1990 patent (U.S. Pat. No. 4,940,782), a murine IgG monoclonal antibody 44.7b specific for rat IgE, which is capable of binding to CD23-bound IgE but not IgE bound to FcεRI, was disclosed. This antibody 44.7b was never pursued as a therapeutic candidate for treating IgE-mediated diseases. A paper published in 2012 (Shiung et al., Immunobiology, 2012, 217:676-683) reported the discovery of a murine anti-human IgE monoclonal antibody, 8D6, which is capable of binding to CD23-bound IgE but not IgE bound to FcεRI. The authors suggested that this antibody possesses not only the major pharmacologic properties of Omalizumab to neutralize IgE without sensitizing mast cells and basophils but also the property of lumiliximab through crosslinking IgE bound to CD23. However, the murine antibody may be immunogenic and cannot be applied for treating IgE-mediated diseases. The murine antibody for therapeutic applications in man has been found to be limited by immune responses made by patients to the murine antibodies. Thus, the humanization of murine antibodies is necessary to reduce immunogenicity in human recipients.